Compositions and methods for nanoparticle lyophile forms

ABSTRACT

This invention provides compositions for making a solid lyophile of one or more nucleic acid active agents, which can be reconstituted as a drug product. The composition can include an aqueous suspension of lipid nanoparticles in a pharmaceutically acceptable solution, wherein the lipid nanoparticles encapsulate one or more nucleic acid active agents, a dextrin compound, and a saccharide compound. The nucleic acid active agents can be RNAi molecules capable of mediating RNA interference, as well as other RNAs and oligonucleotides.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/195,356, filed Jul. 22, 2015, entitled COMPOSITIONS AND METHODSFOR NANOPARTICLE LYOPHILE FORMS, the contents of which is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Therapeutics based on nucleic acid compounds include various RNA formssuch as siRNAs, antisense RNA, microRNAs, as well as various forms ofDNAs and plasmids, hybrid oligonucleotides, and aptamers, among others.

Transfection of nucleic acid therapeutics and other agents has beenaccomplished by encapsulating the active molecules in lipidnanoparticles. Drawbacks of this methodology include the inability tostore compositions for later use because of degradation of thenanoparticles or their encapsulated cargo. For example, compositions oflipid nanoparticles that encapsulate siRNA molecules may be stable foronly a few minutes or hours at 25° C., and only a few days or weeks at4° C. Further drawbacks include the need for very low temperaturestorage of the lipid nanoparticle compositions.

One way to provide for long-term storage of a therapeutic composition isto prepare a lyophile form, which can be stored and reconstituted toprovide a formulation for administration of the therapeutic.

However, it has not been possible in general to generate lyophile formsof lipid nanoparticles containing nucleic acid agents, so that the lipidnanoparticle can be regenerated with the nucleic acid agent encapsulatedto form a stable formulation. The lyophilization process can destroy thenanoparticles and/or the nucleic acid agents. Some methods have involvedchemically attaching protective groups or components to the lipidnanoparticles, or to the nucleic acid agent, which is disadvantageous.Other methods may use liposomes as an adjuvant, without providing forencapsulation of the nucleic acid agents.

There is a continuing need for compositions and methods to providelyophile forms of nanoparticles that can be reconstituted with favorableproperties, including transfection activity, particle size, storagetime, and serum stability to deliver various nucleic acid agents.

What is needed are compositions and compounds for forming stablesolutions or suspensions of lipid nanoparticles that can be stored insolid lyophile forms, where the nanoparticles encapsulate nucleic acidagents.

BRIEF SUMMARY

This invention relates to the fields of biopharmaceuticals andtherapeutics composed of nucleic acid based molecules. Moreparticularly, this invention relates to methods and compositions forlyophile forms of nucleic acid therapeutic compositions.

This invention provides methods and compositions for therapeuticscomposed of nucleic acid based molecules. More particularly, thisinvention provides methods and compositions for lyophile forms ofnucleic acid based therapeutic compositions.

This invention further provides lyophile forms of nanoparticles that canbe reconstituted into effective therapeutic compositions, which can beused to deliver therapeutic nucleic acid agents for transfection.

In some aspects, this invention provides compositions and compounds forforming solutions or suspensions of therapeutic lipid nanoparticles thatare stable in lyophilization processes. The therapeutic lipidnanoparticles can encapsulate nucleic acid agents, and can betransformed and stored in solid lyophile forms. The lyophile forms canbe reconstituted to provide therapeutic lipid nanoparticles withencapsulated nucleic acid agents. The reconstituted lipid nanoparticlescan have surprisingly advantageous transfection properties, includingparticle size and distribution.

Embodiments of this invention include a range of compositions andcompounds for forming solutions or suspensions of therapeutic lipidnanoparticles that can undergo a lyophilization process to providestable, solid lyophile forms for long-term storage of a nucleic acidtherapeutic.

Embodiments of this invention include the following:

A composition for making a solid lyophile of lipid nanoparticlescomprising one or more nucleic acid active agents, the compositioncomprising:

an aqueous suspension of the lipid nanoparticles in a pharmaceuticallyacceptable solution, wherein the lipid nanoparticles encapsulate the oneor more nucleic acid active agents;

a dextrin compound; and

a saccharide sugar compound.

The composition above, wherein the total amount of the dextrin and sugarcompounds is from 2% to 20% (w/v) of the composition.

The composition above, wherein the dextrin compound is from 40% to 70%(w/v) of the total amount of the dextrin and sugar compounds.

The composition above, wherein the dextrin compound is from 40% to 55%(w/v) of the total amount of the dextrin and sugar compounds.

The composition above, wherein the dextrin compound is 40% to 45% (w/v)of the total amount of the dextrin and sugar compounds.

The composition above, wherein upon lyophilization and reconstitution ofthe composition, the average size of the nanoparticles is within 10% oftheir size in the original composition.

The composition above, wherein upon lyophilization, storage andreconstitution of the composition, the average size of the nanoparticlesis within 10% of their size in the original composition.

The composition above, wherein the lyophilized composition is stored at5° C. for at least one month.

The composition above, wherein the lyophilized composition is stored at−20° C. for at least one month.

The composition above, wherein the nanoparticles have an averagediameter of from 45 nm to 110 nm.

The composition above, wherein the concentration of the nucleic acidactive agents is from 1 mg/mL to 10 mg/mL, or from 3 mg/mL to 5 mg/mL.

The composition above, wherein the one or more nucleic acid activeagents are RNAi molecules capable of mediating RNA interference. Thecomposition above, wherein the RNAi molecules are siRNAs, shRNAs,ddRNAs, piRNAs, or rasiRNAs.

The composition above, wherein the one or more nucleic acid activeagents are miRNAs, antisense RNAs, plasmids, hybrid oligonucleotides, oraptamers.

The composition above, wherein the pharmaceutically acceptable solutionis a HEPES buffer, a phosphate buffer, a citrate buffer, or a buffercontaining Tris(hydroxymethyl)aminomethane.

The composition above, wherein the dextrin compound is a cyclodextrin.

The composition above, wherein the cyclodextrin compound has one or moreof the 2, 3 and 6 hydroxyl positions substituted with sulfoalkyl,benzenesulfoalkyl, acetoalkyl, hydroxyalkyl, hydroxyalkyl succinate,hydroxyalkyl malonate, hydroxyalkyl glutarate, hydroxyalkyl adipate,hydroxyalkyl, hydroxyalkyl maleate, hydroxyalkyl oxalate, hydroxyalkylfumarate, hydroxyalkyl citrate, hydroxyalkyl tartrate, hydroxyalkylmalate, or hydroxyalkyl citraconate groups.

The composition above, wherein the cyclodextrin compound is(2-hydroxypropyl)-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrinsuccinate, (2-hydroxypropyl)-γ-cyclodextrin, or2-hydroxypropyl-γ-cyclodextrin succinate.

The composition above, wherein the cyclodextrin compound is sulfobutylether β-cyclodextrin or sulfobutyl ether γ-cyclodextrin.

The composition above, wherein the cyclodextrin compound ismethyl-β-cyclodextrin or methyl-γ-cyclodextrin.

The composition above, wherein the cyclodextrin compound is attached toa polymer chain or network.

The composition above, wherein the cyclodextrin compound includes anadsorbate compound.

The composition above, wherein the adsorbate compound is selected fromcholesterol, lanosterol, zymosterol, zymostenol, desmosterol,stigmastanol, dihydrolanosterol, 7-dehydrocholesterol, pegylatedcholesterol, cholesteryl acetate, cholesteryl arachidonate, cholesterylbutyrate, cholesteryl hexanoate, cholesteryl myristate, cholesterylpalmitate, cholesteryl behenate, cholesteryl stearate, cholesterylcaprylate, cholesteryl n-decanoate, cholesteryl dodecanoate, cholesterylnervonate, cholesteryl pelargonate, cholesteryl n-valerate, cholesteryloleate, cholesteryl elaidate, cholesteryl erucate, cholesterylheptanoate, cholesteryl linolelaidate, cholesteryl linoleate,beta-sitosterol, campesterol, ergosterol, brassicasterol,delta-7-stigmasterol, and delta-7-avenasterol.

The composition above, wherein the saccharide sugar compound is amonosaccharide or disaccharide sugar compound.

The composition above, wherein the sugar compound is selected fromsucrose, lactose, lactulose, maltose, trehalose, cellobiose, kojibiose,sakebiose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose,maltulose, isomaltulose, gentiobiulose, mannobiose, melibiose,melibiulose, and xylobiose.

A process for making a solid lyophile of one or more nucleic acid activeagents, the process comprising lyophilizing a composition describedabove. This invention further contemplates a solid lyophile made by theprocess above, as well as a drug product made by reconstituting a solidlyophile above.

This invention further includes a process for making a nucleic acid drugproduct, the process comprising:

synthesizing lipid nanoparticles, wherein the lipid nanoparticlesencapsulate one or more nucleic acid active agents;

providing an aqueous suspension of the lipid nanoparticles in apharmaceutically acceptable solution;

adding a dextrin compound to the solution containing the lipidnanoparticles;

adding a saccharide sugar compound to the solution containing the lipidnanoparticles;

lyophilizing the solution containing the lipid nanoparticles, therebyforming a solid lyophile;

reconstituting the lyophile in a pharmaceutically acceptable carrier,thereby forming a nucleic acid drug product.

The process above, wherein the total amount of the dextrin andsaccharide sugar compounds is from 2% to 20% (w/v) of the solutioncontaining the lipid nanoparticles.

The process above, wherein the dextrin compound is from 40% to 70% (w/v)of the total amount of the dextrin and saccharide sugar compounds.

The process above, wherein the dextrin compound is from 40% to 55% (w/v)of the total amount of the dextrin and saccharide sugar compounds.

The process above, wherein the dextrin compound is 40% to 45% (w/v) ofthe total amount of the dextrin and saccharide sugar compounds.

The process above, wherein upon reconstitution, the average size of thenanoparticles is within 10% of their size when synthesized.

The process above, further comprising storing the lyophile beforereconstitution.

The process above, wherein upon storage and reconstitution of thelyophile, the average size of the nanoparticles is within 10% of theirsize when synthesized.

The process above, wherein the lyophile is stored at 5° C. for at leastone month.

The process above, wherein the lyophile is stored at −20° C. for atleast one month.

The process above, wherein the nanoparticles have an average diameter offrom 45 nm to 110 nm.

The process above, wherein the concentration of the nucleic acid activeagents is from 1 mg/mL to 10 mg/mL.

The process above, wherein the one or more nucleic acid active agentsare RNAi molecules capable of mediating RNA interference. The processabove, wherein the RNAi molecules are siRNAs, shRNAs, ddRNAs, piRNAs, orrasiRNAs.

The process above, wherein the one or more nucleic acid active agentsare miRNAs, antisense RNAs, plasmids, hybrid oligonucleotides, oraptamers.

The process above, wherein the pharmaceutically acceptable carrier issterile water, water for injection, sterile normal saline,bacteriostatic water for injection, or a nebulizer solution.

The process above, wherein the pharmaceutically acceptable carrier is apharmaceutically acceptable solution.

The process above, wherein the pharmaceutically acceptable solution is aHEPES buffer, a phosphate buffer, a citrate buffer, or a buffercontaining Tris(hydroxymethyl)aminomethane.

The process above, wherein the dextrin compound is a cyclodextrin.

The process above, wherein the cyclodextrin compound has one or more ofthe 2, 3 and 6 hydroxyl positions substituted with sulfoalkyl,benzenesulfoalkyl, acetoalkyl, hydroxyalkyl, hydroxyalkyl succinate,hydroxyalkyl malonate, hydroxyalkyl glutarate, hydroxyalkyl adipate,hydroxyalkyl, hydroxyalkyl maleate, hydroxyalkyl oxalate, hydroxyalkylfumarate, hydroxyalkyl citrate, hydroxyalkyl tartrate, hydroxyalkylmalate, or hydroxyalkyl citraconate groups.

The process above, wherein the cyclodextrin compound is(2-hydroxypropyl)-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrinsuccinate, (2-hydroxypropyl)-γ-cyclodextrin, or2-hydroxypropyl-γ-cyclodextrin succinate.

The process above, wherein the cyclodextrin compound is sulfobutyl ether3-cyclodextrin or sulfobutyl ether γ-cyclodextrin.

The process above, wherein the cyclodextrin compound ismethyl-β-cyclodextrin or methyl-γ-cyclodextrin.

The process above, wherein the cyclodextrin compound includes anadsorbate compound.

The process above, wherein the saccharide sugar compound is amonosaccharide or disaccharide sugar compound.

The process above, wherein the pharmaceutically acceptable carrier issterile water, water for injection, sterile normal saline,bacteriostatic water for injection, or a nebulizer solution.

The process above, wherein the pharmaceutically acceptable carrier is apharmaceutically acceptable solution.

The process above, wherein the reconstituted nucleic acid drug producthas less 0.001% (w/v) of aggregate particles with a size greater than0.2 μm.

The process above, wherein the nucleic acid drug product isreconstituted in a time period of 3 to 30 seconds.

The process above, wherein the nucleic acid drug product isreconstituted after a storage time period of six months and retains 80%activity of the nucleic acid agents.

The process above, wherein the reconstituted nucleic acid drug producthas less 0.001% (w/v) of aggregate particles with a size greater than0.2 μm.

The process above, wherein the reconstituted nucleic acid drug producthas reduced cytokine activation.

The process above, wherein the nucleic acid drug product isreconstituted in a time period of 3 to 30 seconds.

The process above, wherein the nucleic acid drug product isreconstituted after a storage time period of six months and retains 80%activity of the nucleic acid agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows experimental results for in vivo potency of a nucleic acidagent, which was a siRNA targeted to suppress Hsp47 (GP46), obtainedwith a final drug product that was a reconstituted solution of a solid,lyophilized nanoparticle formulation of the siRNA. Reconstituted siRNAdrug formulations were used in a Dimethylnitrosamine (DMN) Induced LiverFibrosis Rat Model. As shown in FIG. 1, the reconstituted siRNAnanoparticle drug formulation exhibited profound and surprising potencyfor gene silencing of Hsp47 (GP46) in vivo. The in vivo potency is arigorous test for the viability of lyophilized, reconstitutednanoparticles containing a nucleic acid agent. The nanoparticleformulation of the siRNA that was lyophilized included a totalprotectant content of 10% (w/v), which was composed of 40% (w/v)(2-hydroxypropyl)-β-cyclodextrin and 60% sucrose.

FIG. 2 shows experimental results for plasma concentrationpharmacokinetics in vivo of a lyophilized, reconstituted siRNAnanoparticle formulation. A siRNA targeted to Hsp47 (GP46) wasformulated in liposomal nanoparticles. The nanoparticle formulationswere lyophilized with a protectant composition containing sucrose and(2-hydroxypropyl)-β-cyclodextrin. The nanoparticle formulation of thesiRNA that was lyophilized included a total protectant content of 12.5%(w/v), which was composed of 40% (w/v) (2-hydroxypropyl)-β-cyclodextrinand 60% sucrose. Plasma PK profiles were evaluated in Sprague Dawleyrats following an intravenous administration at a single dose level ofthe lyophilized formulation compared to a frozen formulation. siRNAconcentrations in plasma samples were determined by ahybridization-based ELISA method. As shown in FIG. 2, the plasmaconcentration pharmacokinetics of the lyophilized, reconstituted siRNAdrug formulation was essentially the same as a comparative controlformulation that had only been frozen.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods and compositions for therapeuticscomposed of nucleic acid based molecules. In some embodiments, thisinvention provides methods and compositions for making lyophile forms oftherapeutic compositions containing nucleic acid agents.

In some aspects, this invention provides lyophile forms of nanoparticlesthat can be reconstituted into effective therapeutic compositions. Thenanoparticles can encapsulate nucleic acid agents as cargo. The lyophileforms of this invention can be used to re-compose and delivernanoparticle formulations encapsulating therapeutic nucleic acid agentsfor transfection.

In further aspects, this invention provides compounds and methods forforming solutions or suspensions of therapeutic lipid nanoparticles thatare stable in lyophilization processes. The lyophilization processes ofthis invention can provide stable lyophile forms of therapeutic lipidnanoparticles, in which the nanoparticles can encapsulate nucleic acidagents. The lyophile forms can be stored for a period of time, andreconstituted to provide therapeutic lipid nanoparticles withencapsulated nucleic acid agents.

In some embodiments, this invention includes a range of compositions andcompounds for solutions or suspensions of lipid nanoparticles that canundergo a lyophilization process to provide stable, solid lyophile formsfor long-term storage of a nucleic acid therapeutic. Compositions andprocesses of this invention can provide lyophile forms that can bereconstituted and provide advantageous activity, particle size, storagetime, and serum stability.

In further aspects, this invention relates to compounds, compositionsand methods for providing nanoparticles to deliver and distribute activeagents or drug compounds to subjects, tissues, and organs.

This invention provides a range of lipid compounds and ionizablecompounds for delivering active agents to cells. The lipid compounds andionizable compounds of this disclosure can be used to form nanoparticlesto deliver and distribute active agents.

This invention contemplates lipid nanoparticle drug formulationscontaining, for example, siRNA agents, which can be prepared bylyophilization of a suspension of the nanoparticles, and reconstitutionof the nanoparticles into a suspension.

In some embodiments, lipid nanoparticles can be synthesized by highspeed injection of lipid/ethanol solution into an siRNA buffer solution.A second buffer can be diafiltered and used as an external bufferthrough TFF cartridges to make a final product aqueous suspension.

In some embodiments, the nanoparticles can have an average diameter offrom 45 nm to 110 nm. The concentration of the nucleic acid activeagents can be from 1 mg/mL to 10 mg/mL.

It was surprisingly found that lipid nanoparticles can survivelyophilization of the suspension, when the suspension is made into aprotected composition.

In some embodiments, a protected composition of this invention can becomposed of an aqueous suspension of the lipid nanoparticles in apharmaceutically acceptable solution, a dextrin compound, and asaccharide sugar compound. The lipid nanoparticles can encapsulate anactive agent, such as one or more nucleic acid active agents.

Lyophilization of the protected suspension can provide a solid lyophileproduct, which can be reconstituted into a suspension of lipidnanoparticles.

The reconstituted suspension can contain lipid nanoparticles, whichencapsulate the active agent and are comparable to the lipidnanoparticles before lyophilization.

In certain embodiments, the reconstituted suspension can provideactivity of the encapsulated agent, which is comparable to that of thesuspension before lyophilization.

In further aspects, the reconstituted suspension can provide stablenanoparticles comparable to that of the suspension beforelyophilization. In certain aspects, the average particle size of thenanoparticles can be nearly equal to the size of the nanoparticles inthe suspension before lyophilization.

The compositions and processes of this invention can provide surprisingactivity and stability of a reconstituted suspension composed ofnanoparticles having an encapsulated agent.

In further aspects, the protected suspension, which can be lyophilizedand reconstituted, can contain a protectant composition forlyophilization. A protectant composition of this invention can becomposed of a dextrin compound and a saccharide sugar compound. Thetotal amount of the dextrin and sugar compounds may be from 2% to 20%(w/v) of the protected suspension.

In some embodiments, the dextrin compound can be from 40% to 70% (w/v)of the total amount of the dextrin and sugar compounds in the protectantcomposition. In certain embodiments, the dextrin compound can be from40% to 55% (w/v) of the total amount of the dextrin and sugar compoundsin the protectant composition. In further embodiments, the dextrincompound may be from 40% to 45% (w/v) of the total amount of the dextrinand sugar compounds in the protectant composition. These compositionscan provide unexpectedly advantageous properties of a reconstitutednanoparticle suspension, for example, insignificant change of thenanoparticle size or activity.

In some aspects, upon lyophilization and reconstitution of a protectedsuspension of nanoparticles, the average size of the nanoparticles canbe within 10% of their size in the original composition, beforelyophilization. In certain aspects, upon lyophilization andreconstitution of a protected suspension of nanoparticles, the averagesize of the nanoparticles can be within 5% of their size in the originalcomposition, before lyophilization.

This invention contemplates lipid nanoparticle drug formulationscontaining, for example, siRNA agents, which can be prepared bylyophilization of a suspension of the nanoparticles, and reconstitutionof the nanoparticles into a suspension after a period of storage. Thereconstituted suspension can provide activity of the encapsulated agent,which is comparable to that of the suspension before lyophilization.

The reconstituted suspension, prepared after a period of storage, cancontain lipid nanoparticles, which encapsulate the active agent and arecomparable to the lipid nanoparticles before lyophilization.

In certain embodiments, the reconstituted suspension, prepared after aperiod of storage, can provide activity of the encapsulated agent, whichis comparable to that of the suspension before lyophilization.

In further aspects, the reconstituted suspension, prepared after aperiod of storage, can provide stable nanoparticles comparable to thatof the suspension before lyophilization. In certain aspects, the averageparticle size of the nanoparticles can be nearly equal to the size ofthe nanoparticles in the suspension before lyophilization.

In some embodiments, the lyophilized composition can be stored at 5° C.for at least one month. In further embodiments, the lyophilizedcomposition can be stored at −20° C. for at least one month.

Active Agents

The compositions and methods of this invention can be used to distributeagents for suppressing gene expression. Examples of an agent forsuppressing gene expression include inhibitory nucleic acid molecules,including ribozymes, anti-sense nucleic acids, and RNA interferencemolecules (RNAi molecules).

Therapeutic compositions of this invention can include inhibitorynucleic acid molecules. Examples of nucleic acid molecules capable ofmediating RNA interference include molecules active in RNA interference(RNAi molecules), including a duplex RNA such as an siRNA (smallinterfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA(DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeatassociated siRNA), and modified forms thereof.

Examples of active therapeutics of this invention include DNAs,plasmids, hybrid oligonucleotides, or aptamers.

The concentration of the active nucleic acid molecules in apre-lyophilization formulation of this disclosure can be from about 1mg/mL to about 10 mg/mL. In some embodiments, the concentration of theactive nucleic acid molecules in a formulation of this disclosure can befrom about 1 mg/mL to about 5 mg/mL, or from 2 mg/mL to 4 mg/mL.

Pre-Lyophilization Lipid Nanoparticle Formulations

Embodiments of this invention can provide compositions of lipidnanoparticles, which compositions contain a protectant compound for alyophilization process.

The lipid nanoparticles can have any composition known in the art. Thelipid nanoparticles may be synthesized and loaded with encapsulatedcargo by any process, including processes known in the art.

In some embodiments, the lipid nanoparticles can be prepared by asubmersion injection process. Some examples of processes for lipidnanoparticles are given in US 2013/0115274.

Some examples for preparing liposomes are given in Szoka, Ann. Rev.Biophys. Bioeng. 9:467 (1980); Liposomes, Marc J. Ostro, ed., MarcelDekker, Inc., New York, 1983, Chapter 1.

In general, lipid nanoparticles can be synthesized by mixing lipidcomponents in an organic solvent with an aqueous buffer solutioncontaining active nucleic acid agents. The liposomes can be sized byfiltration or extrusion. The liposome suspension or solution may befurther transformed by diafiltration.

A lipid nanoparticle composition of this invention, which is stabilizedfor a lyophilization process, may contain lipid nanoparticles thatencapsulate one or more active agents, such as nucleic acid agents, in asuspension. The suspension can be aqueous, and may contain awater-miscible solvent, such as ethanol. The composition, which isstabilized for a lyophilization process, may further contain protectantcompounds to stabilize the liposomes in the lyophilization process.

The average size of lipid nanoparticles as synthesized can be from 40 nmto 120 nm, or from 45 nm to 110 nm, or from 85 nm to 105 nm.

The concentration of the active agent in a lipid nanoparticlecomposition of this invention can range from about 0.1 mg/mL to about 10mg/mL. In some embodiments, the concentration of the active agent in alipid nanoparticle composition of this invention can be from 0.5 mg/mLto 8 mg/mL, or from 1 mg/mL to 6 mg/mL, or from 2 mg/mL to 5 mg/mL, orfrom 3 mg/mL to 4 mg/mL.

Examples of protectant compounds include dextrin compounds.

Examples of dextrin compounds include maltodextrins, and beta- andgamma-cyclodextrins.

Examples of dextrin compounds include methylated beta- andgamma-cyclodextrin compounds, and sulfoalkyl ether beta- andgamma-cyclodextrin compounds.

Examples of dextrin compounds include cyclodextrin compounds having oneor more of the 2, 3 and 6 hydroxyl positions substituted withsulfoalkyl, benzenesulfoalkyl, acetoalkyl, hydroxyalkyl, hydroxyalkylsuccinate, hydroxyalkyl malonate, hydroxyalkyl glutarate, hydroxyalkyladipate, hydroxyalkyl, hydroxyalkyl maleate, hydroxyalkyl oxalate,hydroxyalkyl fumarate, hydroxyalkyl citrate, hydroxyalkyl tartrate,hydroxyalkyl malate, or hydroxyalkyl citraconate groups.

Examples of dextrin compounds include (2-hydroxypropyl)-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin succinate,(2-hydroxypropyl)-γ-cyclodextrin, and 2-hydroxypropyl-γ-cyclodextrinsuccinate.

Examples of dextrin compounds include hydroxyethyl β-cyclodextrin.

Examples of dextrin compounds include dimethyl β-cyclodextrin andtrimethyl β-cyclodextrin.

Examples of dextrin compounds include sulfobutyl ether β-cyclodextrinand sulfobutyl ether γ-cyclodextrin.

Examples of dextrin compounds include methyl-β-cyclodextrin andmethyl-γ-cyclodextrin.

Examples of dextrin compounds includehydroxypropyl-sulfobutyl-β-cyclodextrin.

Examples of dextrin compounds include H107 SIGMA cyclodextrin(Sigma-Aldrich Corp.).

Examples of dextrin compounds include CAVAMAX, CAVASOL, and CAVATRONcyclodextrins (Ashland Inc.).

Examples of dextrin compounds include KLEPTOSE and CRYSMEB cyclodextrins(Roquette America Inc.).

Examples of dextrin compounds include CAPTISOL cyclodextrins (LigandPharmaceuticals, Inc.).

In some embodiments, examples of dextrin compounds include dextrincompounds attached to a polymer chain or network. For example,cyclodextrin molecules can be attached to polymers of polyacrylic acid.In further embodiments, cyclodextrin molecules can be linked togetherwith cross linking compounds such as acryloyl groups. In certainembodiments, vinyl acrylate hydrogel forms with attached cyclodextrincompounds can be used.

In some aspects, a dextrin compound to be used in a lipid nanoparticlecomposition of this invention can be combined with an adsorbate compoundbefore being introduced into the lipid nanoparticle composition. Withoutwishing to be bound by any one particular theory, the pre-adsorption ofa sterol compound by the dextrin compound may form an inclusion complexthat can prevent a loss of activity of the active agent in thereconstituted drug product.

Examples of adsorbate compounds include cholesterol, lanosterol,zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol,7-dehydrocholesterol.

Examples of adsorbate compounds include pegylated cholesterols, andcholestane 3-oxo-(C1-22)acyl compounds, for example, cholesterylacetate, cholesteryl arachidonate, cholesteryl butyrate, cholesterylhexanoate, cholesteryl myristate, cholesteryl palmitate, cholesterylbehenate, cholesteryl stearate, cholesteryl caprylate, cholesteryln-decanoate, cholesteryl dodecanoate, cholesteryl nervonate, cholesterylpelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesterylelaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryllinolelaidate, and cholesteryl linoleate.

Examples of adsorbate compounds include phytosterols, beta-sitosterol,campesterol, ergosterol, brassicasterol, delta-7-stigmasterol, anddelta-7-avenasterol.

Additional examples of protectant compounds include saccharidecompounds. Examples of saccharide compounds include sugar compounds.

Examples of protectant sugar compounds include monosaccharides such asC(5-6) aldoses and ketoses, as well as disaccharides such as sucrose,lactose, lactulose, maltose, trehalose, cellobiose, kojibiose,sakebiose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose,maltulose, isomaltulose, gentiobiulose, mannobiose, melibiose,melibiulose, and xylobiose.

Examples of protectant saccharide compounds include polysaccharides suchas ficoll.

The concentration of protectant compounds in the pre-lyophilizationformulation can be from about 1% (w/v) to about 25% (w/v).

In some embodiments, the concentration of protectant compounds in thepre-lyophilization formulation can be from 2% (w/v) to 20% (w/v), orfrom 4% (w/v) to 16% (w/v), or from 5% (w/v) to 15% (w/v), or from 6%(w/v) to 14% (w/v), or from 8% (w/v) to 12% (w/v).

In certain embodiments, the concentration of protectant compounds in thepre-lyophilization formulation can be 6% (w/v), or 8% (w/v), or 10%(w/v), or 12% (w/v), or 14% (w/v), or 16% (w/v), or 18% (w/v), or 20%(w/v), or 22% (w/v), or 24% (w/v).

Lyophilization Processes

Lyophilization processes can be carried out in any suitable vessel, suchas glass vessels, or, for example, glass vials, or dual-chamber vessels,as are known in the pharmaceutical arts.

A stabilized lipid nanoparticle composition of this invention containinga protectant compound can be introduced into to the glass vessel. Thevolume of the composition added to the vessel can be from 0.1-20 mL, orfrom 1-10 mL.

Any lyophilization process can be used, including those known in thepharmaceutical arts. See, e.g., Remington's Pharmaceutical Sciences,18th Ed., Mack Publishing Co., Easton, Penn. (1990).

The lyophilization process can include freezing theprotectant-stabilized lipid nanoparticle composition at a temperature offrom about −40° C. to about −30° C. The frozen composition can be driedform a lyophilized composition.

In some embodiments, the freezing step can ramp the temperature fromambient to the final temperature over several minutes. The temperatureramp can be about 1° C./minute.

In some embodiments, the drying step can be performed at a pressure ofabout 0-250 mTorr, or 50-150 mTorr, at a temperature of from about −15°C. to about −38° C. The drying step can be continued at a highertemperature, up to ambient temperature, over a period of up to severaldays. The level of residual water in the solid lyophile can be less thanabout 5%, or less than 4%, or less than 3%, or less than 2%, or lessthan 1% (w/v).

The protectant-stabilized lipid nanoparticle compositions of thisinvention, after lyophilization, can be reconstituted by methods knownin the pharmaceutical arts.

In some aspects, this invention provides methods for inhibiting thelevel of aggregated particles in a reconstituted drug product, made froma protectant-stabilized lipid nanoparticle composition of this inventionafter lyophilization.

In some embodiments, the reconstituted drug product, made from aprotectant-stabilized lipid nanoparticle composition of this inventionafter lyophilization, can have reduced levels of aggregate particles.

In certain embodiments, the reconstituted drug product, made from aprotectant-stabilized lipid nanoparticle composition of this inventionafter lyophilization, can have reduced levels of aggregate particleswith a size greater than about 0.2 μm, or greater than about 0.5 μm, orgreater than about 1 μm.

Reconstituted Drug Product

The lyophile can be reconstituted in a pharmaceutically acceptablecarrier.

Examples of a pharmaceutically acceptable carrier include sterile water,water for injection, sterile normal saline, bacteriostatic water forinjection, and a nebulizer solution.

Examples of a pharmaceutically acceptable carrier include apharmaceutically acceptable solution.

Examples of a pharmaceutically acceptable solution include HEPES buffer,phosphate buffers, citrate buffers, and a buffer containingTris(hydroxymethyl)aminomethane.

Examples of a pharmaceutically acceptable solutions includepharmaceutically acceptable buffer solutions.

Examples of a pharmaceutically acceptable solution include buffersolutions of maleic acid, tartaric acid, lactic acid, acetic acid,sodium bicarbonate, and glycine.

The reconstituted lyophile can be used as a drug product.

The reconstituted lyophile can be further diluted with isotonic salineor other excipients to provide a predetermined concentration foradministration.

Examples of excipients include tonicifiers.

Examples of excipients include stabilizers such as human serum albumin,bovine serum albumin, a-casein, globulins, a-lactalbumin, LDH, lysozyme,myoglobin, ovalbumin, and RNase A.

Examples of excipients include buffers such as potassium acetate, sodiumacetate, and sodium bicarbonate.

Examples of excipients include amino acids such as glycine, alanines,arginine, betaine, leucine, lysine, glutamic acid, aspartic acid,histidine, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid,alanopine, octopine, strombine, and trimethylamine N-oxide.

Examples of excipients include non-ionic surfactants such as polysorbate20, polysorbate 80, and poloxamer 407.

Examples of excipients include dispersing agents such as phosphotidylcholine, ethanolamine, acethyltryptophanate, polyethylene glycol,polyvinylpyrrolidone, ethylene glycol, glycerin, glycerol, propyleneglycol, sorbitol, xylitol, dextran, and gelatin.

Examples of excipients include antioxidants such as ascorbic acid,cysteine, thioglycerol, thioglycolic acid, thiosorbitol, andglutathione.

Examples of excipients include reducing agents such as dithiothreitol,thiols, and thiophenes.

Examples of excipients include chelating agents such as EDTA, EGTA,glutamic acid, and aspartic acid.

In some embodiments, the lyophile can be reconstituted using a syringeneedle through a stoppered vial. The lyophile can be reconstituted withor without shaking the vial.

The time for reconstitution can be from 3-30 seconds, or longer.

In some embodiments, the reconstituted nucleic acid drug product canhave less than 0.001% (w/v) of aggregate particles with a size greaterthan 0.2 μm.

In certain aspects, the reconstituted nucleic acid drug product can havereduced cytokine activation.

In additional aspects, the nucleic acid drug product can bereconstituted after a storage time period of six months and retain 80%activity of the nucleic acid agents.

In some embodiments, the nucleic acid drug product can be reconstitutedafter a storage time period of six months and the average particle sizeof the lipid nanoparticles can be less than 25% greater than beforelyophilization.

In certain embodiments, the nucleic acid drug product can bereconstituted after a storage time period of 24 months and retain 90%activity of the nucleic acid agents.

In further embodiments, the nucleic acid drug product can bereconstituted after a storage time period of 24 months and the averageparticle size of the lipid nanoparticles can be less than 25% greaterthan before lyophilization.

RNAi Molecules

The amount of active RNA interference inducing ingredient formulated inthe composition of the present invention may be an amount that does notcause an adverse effect exceeding the benefit of administration. Such anamount may be determined by an in vitro test using cultured cells, or atest in a model animal or mammal such as a mouse, a rat, a dog, or apig, etc., and such test methods are known to those skilled in the art.The methods of this invention can be applicable to any animal, includinghumans.

The amount of active ingredient formulated can vary according to themanner in which the agent or composition is administered. For example,when a plurality of units of the composition is used for oneadministration, the amount of active ingredient to be formulated in oneunit of the composition may be determined by dividing the amount ofactive ingredient necessary for one administration by said plurality ofunits.

The nucleic acid molecules and RNAi molecules of this invention can bedelivered or administered to a cell, tissue, organ, or subject by directapplication of the molecules in liposome formulations to assist, promoteor facilitate entry into a cell.

The nucleic acid molecules and RNAi molecules of this invention can becomplexed with cationic lipids, packaged within liposomes, and deliveredto target cells or tissues. The nucleic acid or nucleic acid complexescan be locally administered to relevant tissues ex vivo, or in vivothrough direct dermal application, transdermal application, orinjection.

A inhibitory nucleic acid molecule or composition of this invention maybe administered in unit dosage form. Conventional pharmaceuticalpractice may be employed to provide suitable formulations orcompositions to administer the compounds to patients suffering from adisease. Any appropriate route of administration may be employed, forexample, administration may be parenteral, intravenous, intraarterial,subcutaneous, intratumoral, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal,intracistemal, intraperitoneal, intranasal, aerosol, suppository, ororal administration.

Compositions and methods of this disclosure can include an expressionvector that includes a nucleic acid sequence encoding at least one RNAimolecule of this invention in a manner that allows expression of thenucleic acid molecule.

The nucleic acid molecules and RNAi molecules of this invention can beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors.

For example, the vector may contain sequences encoding both strands of aRNAi molecule of a duplex, or a single nucleic acid molecule that isself-complementary and thus forms a RNAi molecule. An expression vectormay include a nucleic acid sequence encoding two or more nucleic acidmolecules.

A nucleic acid molecule may be expressed within cells from eukaryoticpromoters. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector.

Lipid formulations can be administered to animals by intravenous,intramuscular, or intraperitoneal injection, or orally or by inhalationor other methods as are known in the art.

Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used.

In one embodiment of the above method, the inhibitory nucleic acidmolecule is administered at a dosage of about 5 to 500 mg/m²/day, e.g.,5, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/m²/day.

In some embodiments, the inhibitory nucleic acid molecules of thisinvention are administered systemically in dosages from about 1 to 100mg/kg, e.g., 1, 5, 10, 20, 25, 50, 75, or 100 mg/kg.

In further embodiments, the dosage can range from about 25 to 500mg/m²/day.

Methods known in the art for making formulations are found, for example,in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro,Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for inhibitory nucleicacid molecules include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for aneoplastic disease or condition. The preferred dosage of a nucleotideoligomer of the invention can depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

Examples of Lipid Compositions

In certain embodiments, the four lipid-like components, i.e. one or moreionizable lipid molecules, a structural lipid, one or more stabilizerlipids, and one or more lipids for reducing immunogenicity of thecomposition, can be 100% of the lipid components of the composition.

Examples of lipid nanoparticle compositions are shown in Table 1.

TABLE 1 Compositions of lipid components (each in mol % of total) ReduceIonizable Cationic Structural Stabilizer immun. 17 0 35 40 8 20 0 35 405 25 0 35 39 1 25 0 35 35 5 25 0 30 40 5 25 0 40 30 5 30 0 25 40 5 35 025 35 5 40 0 30 25 5 25 5 30 35 5 25 10 30 30 5 25 15 25 30 5

Ionizable Lipid-Like Molecules

Examples of an ionizable molecule include compounds having the structureshown in Formula I

wherein R¹ and R² are

R¹═CH₂(CH₂)_(n)OC(═O)R⁴

R²═CH₂(CH₂)_(m)OC(═O)R⁵

-   wherein n and m are each independently from 1 to 2; and R⁴ and R⁵    are independently for each occurrence a C(12-20) alkyl group, or a    C(12-20) alkenyl group;-   wherein R³ is selected from 1-azetidines, 1-pyrrolidines,    1-piperidines, 4-morpholines, and 1,4-piperazines wherein the rings    can be substituted at any carbon atom position,

and can also be selected from amino and aminoalkyl groups, which may besubstituted,

wherein

-   each R⁶ is independently selected from H, alkyl, hydroxyl,    hydroxyalkyl, alkoxy, alkoxyalkoxy, and aminoalkyl;-   each R⁷ is independently selected from H, alkyl, hydroxyalkyl, and    aminoalkyl;-   each R⁸ is independently selected from H, alkyl, hydroxyalkyl, and    aminoalkyl, and any two R⁸ may form a ring;-   q is from zero to four;-   Q is O or NR′;-   p is from 1 to 4.

Examples of on ionizable lipid include the following compound:

which is((2-((3S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of on ionizable lipid include the following compound:

which is((2-(3-(hydroxymethyl)azetidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Examples of on ionizable lipid include the following compound:

which is((2-(4-(2-hydroxyethyl)piperazin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of on ionizable lipid include the following compound:

which is((2-(4-(2-hydroxyethyl)piperazin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of on ionizable lipid include the following compound:

which is2-((1-(((9Z,12Z)-heptadeca-9,12-dien-1-yl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-1,5-dioxopentan-3-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium.

Examples of on ionizable lipid include the following compound:

which is2-((9Z,12Z)—N-(3-(dimethylamino)propyl)octadeca-9,12-dienamido)ethyl(9Z,12Z)-octadeca-9,12-dienoate.

Examples of on ionizable lipid include the following compound:

which isN,N,N-trimethyl-3-((9Z,12Z)—N-(2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)ethyl)octadeca-9,12-dienamido)propan-1-aminium.

Examples of on ionizable lipid include the following compound:

which isN,N,N-trimethyl-2-(((S)-3-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-2-((9Z,12Z)-octadeca-9,12-dienamido)-3-oxopropyl)amino)-2-oxoethan-1-aminium.

Structural Lipids

Examples of structural lipids include cholesterols, sterols, andsteroids.

Examples of structural lipids include cholanes, cholestanes, ergostanes,campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes,gonanes, estranes, androstanes, pregnanes, and cycloartanes.

Examples of structural lipids include sterols and zoosterols such ascholesterol, lanosterol, zymosterol, zymostenol, desmosterol,stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol.

Examples of structural lipids include pegylated cholesterols, andcholestane 3-oxo-(C1-22)acyl compounds, for example, cholesterylacetate, cholesteryl arachidonate, cholesteryl butyrate, cholesterylhexanoate, cholesteryl myristate, cholesteryl palmitate, cholesterylbehenate, cholesteryl stearate, cholesteryl caprylate, cholesteryln-decanoate, cholesteryl dodecanoate, cholesteryl nervonate, cholesterylpelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesterylelaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryllinolelaidate, and cholesteryl linoleate.

Examples of structural lipids include sterols such as phytosterols,beta-sitosterol, campesterol, ergosterol, brassicasterol,delta-7-stigmasterol, and delta-7-avenasterol.

Stabilizer Lipids

Examples of stabilizer lipids include zwitterionic lipids.

Examples of stabilizer lipids include compounds such as phospholipids.

Examples of phospholipids include phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and ordilinoleoylphosphatidylcholine.

Examples of stabilizer lipids include phosphatidyl ethanolaminecompounds and phosphatidyl choline compounds.

Examples of stabilizer lipids include1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC).

Examples of stabilizer lipids include diphytanoyl phosphatidylethanolamine (DPhPE) and 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine(DPhPC).

Examples of stabilizer lipids include1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

Examples of stabilizer lipids include 1,2-dilauroyl-sn-glycerol (DLG);1,2-dimyristoyl-sn-glycerol (DMG); 1,2-dipalmitoyl-sn-glycerol (DPG);1,2-distearoyl-sn-glycerol (DSG);1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC);1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (DPePC);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-Lyso-PC); and1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-Lyso-PC).

Lipids for Reducing Immunogenicity

Examples of lipids for reducing immunogenicity include polymericcompounds and polymer-lipid conjugates.

Examples of lipids for reducing immunogenicity include pegylated lipidshaving polyethyleneglycol (PEG) regions. The PEG regions can be of anymolecular mass. In some embodiments, a PEG region can have a molecularmass of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500,4000 or 5000 Da.

Examples of lipids for reducing immunogenicity include compounds havinga methoxypolyethyleneglycol region.

Examples of lipids for reducing immunogenicity include compounds havinga carbonyl-methoxypolyethyleneglycol region.

Examples of lipids for reducing immunogenicity include compounds havinga multi-branched PEG region.

Examples of lipids for reducing immunogenicity include compounds havinga polyglycerine region.

Examples of lipids for reducing immunogenicity include polymeric lipidssuch as DSPE-mPEG, DMPE-mPEG, DPPE-mPEG, and DOPE-mPEG.

Examples of lipids for reducing immunogenicity include PEG-phospholipidsand PEG-ceramides.

Cationic Lipids

Examples of cationic lipids include HEDC compounds described in US2013/022665 A1, and other compounds described in US 2013/0330401 A1 andUS 2013/0115274 A1. Additional examples of cationic lipids are known inthe art.

Nanoparticle Formulations

Embodiments of this invention can provide liposome nanoparticlecompositions.

In certain embodiments, an ionizable molecule of this invention can beused to form liposome compositions, which can have a bilayer oflipid-like molecules.

A nanoparticle composition can have one or more of the ionizablemolecules of this invention in a liposomal structure, a bilayerstructure, a micelle, a lamellar structure, or a mixture thereof.

In some embodiments, a composition can include one or more liquidvehicle components. A liquid vehicle suitable for delivery of activeagents of this invention can be a pharmaceutically acceptable liquidvehicle. A liquid vehicle can include an organic solvent, or acombination of water and an organic solvent.

Embodiments of this invention can provide lipid nanoparticles having asize of from 10 to 1000 nm. In some embodiments, the liposomenanoparticles can have a size of from 10 to 150 nm.

In certain embodiments, the liposome nanoparticles of this invention canencapsulate the RNAi molecule and retain at least 80% of theencapsulated RNAi molecules after 1 hour exposure to human serum. Thisinvention can provide a composition for use in distributing an activeagent in cells, tissues or organs, organisms, and subjects, where thecomposition includes one or more ionizable lipid molecules of thisinvention.

Compositions of this invention may include one or more of the ionizablelipid molecules, along with a structural lipid, one or more stabilizerlipids, and one or more lipids for reducing immunogenicity of thecomposition.

An ionizable lipid molecule of this invention can be any mol % of acomposition of this invention.

The ionizable lipid molecules of a composition of this invention can befrom 15 mol % to 40 mol % of the lipid components of the composition. Incertain embodiments, the ionizable lipid molecules of a composition canbe from 20 mol % to 35 mol % of the lipid components of the composition.In further embodiments, the ionizable lipid molecules of a compositioncan be from 25 mol % to 30 mol % of the lipid components of thecomposition.

The structural lipid of a composition of this invention can be from 25mol % to 40 mol % of the lipid components of the composition. In certainembodiments, the structural lipid of a composition can be from 30 mol %to 35 mol % of the lipid components of the composition.

The sum of the stabilizer lipids of a composition of this invention canbe from 25 mol % to 40% mol % of the lipid components of thecomposition. In certain embodiments, the sum of the stabilizer lipids ofa composition can be from 30 mol % to 40 mol % of the lipid componentsof the composition.

In some embodiments, a composition of this invention can include two ormore stabilizer lipids, where each of the stabilizer lipids individuallycan be from 5 mol % to 35 mol % of the lipid components of thecomposition. In certain embodiments, a composition of this invention caninclude two or more stabilizer lipids, where each of the stabilizerlipids individually can be from 10 mol % to 30 mol % of the lipidcomponents of the composition.

In certain embodiments, the sum of the one or more stabilizer lipids canbe from 25 mol % to 40 mol % of the lipids of the composition, whereineach of the stabilizer lipids individually can be from 5 mol % to 35%mol %.

In certain embodiments, the sum of the one or more stabilizer lipids canbe from 30 mol % to 40 mol % of the lipids of the composition, whereineach of the stabilizer lipids individually can be from 10 mol % to 30%mol %.

The one or more lipids for reducing immunogenicity of the compositioncan be from a total of 1 mol % to 8 mol % of the lipid components of thecomposition. In certain embodiments, the one or more lipids for reducingimmunogenicity of the composition can be from a total of 1 mol % to 5mol % of the lipid components of the composition.

In additional aspects, a composition of this invention can furtherinclude a cationic lipid, which can be from 5 mol % to 25 mol % of thelipid components of the composition. In certain embodiments, acomposition of this invention can further include a cationic lipid,which can be from 5 mol % to 15 mol % of the lipid components of thecomposition. In these aspects, the molar ratio of the concentrations ofthe cationic lipid to the ionizable lipid molecules of a composition ofthis invention can be from 5:35 to 25:15.

In compositions of this invention, the entirety of the lipid componentsmay include one or more of the ionizable lipid molecular components, oneor more structural lipids, one or more stabilizer lipids, and one ormore lipids for reducing immunogenicity of the composition.

In some embodiments, a composition can contain the ionizable lipidcompound A6, the structural lipid cholesterol, the stabilizer lipidsDOPC and DOPE, and the lipid for reducing immunogenicity DPPE-mPEG. Incertain embodiments, compound A6 can be 15 to 25 mol % of thecomposition; the cholesterol, DOPC, and DOPE combined can be 75 to 85mol % of the composition; and DPPE-mPEG can be 5 mol % of thecomposition.

In one embodiment, compound A6 can be 25 mol % of the composition;cholesterol can be 30 mol % of the composition, DOPC can be 20 mol % ofthe composition, DOPE can be 20 mol % of the composition; andDPPE-mPEG(2000) can be 5 mol % of the composition.

Pharmaceutical Compositions

This invention further contemplates methods for distributing an activeagent to an organ of a subject for treating disease by administering tothe subject a composition of this invention. Organs that can be treatedinclude lung, liver, pancreas, kidney, colon, bone, skin, and intestine.

In further aspects, this invention provides a range of pharmaceuticalformulations.

A pharmaceutical formulation herein can include an active agent, as wellas a drug carrier, or a lipid of this invention, along with apharmaceutically acceptable carrier or diluent. In general, activeagents of this description include any active agents for malignanttumor, including any inhibitory nucleic acid molecules and any smallmolecular drugs. Examples of inhibitory nucleic acid molecules includeribozymes, anti-sense nucleic acids, and RNA interference molecules(RNAi molecules).

A pharmaceutical formulation of this invention may contain one or moreof each of the following: a surface active agent, a diluent, anexcipient, a preservative, a stabilizer, a dye, and a suspension agent.

Some pharmaceutical carriers, diluents and components for apharmaceutical formulation, as well as methods for formulating andadministering the compounds and compositions of this invention aredescribed in Remington's Pharmaceutical Sciences, 18th Ed., MackPublishing Co., Easton, Penn. (1990).

Examples of preservatives include sodium benzoate, ascorbic acid, andesters of p-hydroxybenzoic acid.

Examples of surface active agents include alcohols, esters, sulfatedaliphatic alcohols.

Examples of excipients include sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium metasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, and calcium carboxymethyl cellulose.

Examples of suspension agents include coconut oil, olive oil, sesameoil, peanut oil, soya, cellulose acetate phthalate,methylacetate-methacrylate copolymer, and ester phthalates.

A therapeutic formulation of this invention for the delivery of one ormore molecules active for gene silencing can be administered to a mammalin need thereof. A therapeutically effective amount of the formulationand active agent, which may be encapsulated in a liposome, can beadministered to a mammal for preventing or treating malignant tumor.

The route of administration may be local or systemic.

A therapeutically-effective formulation of this invention can beadministered by various routes, including intravenous, intraperitoneal,intramuscular, subcutaneous, and oral.

Routes of administration may include, for example, parenteral delivery,including intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intranasal, or intraocular injections.

The formulation can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate.

The composition of the present invention may be administered via variousroutes including both oral and parenteral routes, and examples thereofinclude, but are not limited to, oral, intravenous, intramuscular,subcutaneous, local, intrapulmonary, intra-airway, intratracheal,intrabronchial, nasal, rectal, intraarterial, intraportal,intraventricular, intramedullar, intra-lymph-node, intralymphatic,intrabrain, intrathecal, intracerebroventricular, transmucosal,percutaneous, intranasal, intraperitoneal, and intrauterine routes, andit may be formulated into a dosage form suitable for each administrationroute. Such a dosage form and formulation method may be selected asappropriate from any known dosage forms and methods. See e.g. HyojunYakuzaigaku, Standard Pharmaceutics, Ed. by Yoshiteru Watanabe et al.,Nankodo, 2003.

Examples of dosage forms suitable for oral administration include, butare not limited to, powder, granule, tablet, capsule, liquid,suspension, emulsion, gel, and syrup, and examples of the dosage formsuitable for parenteral administration include injections such as aninjectable solution, an injectable suspension, an injectable emulsion,and a ready-to-use injection. Formulations for parenteral administrationmay be a form such as an aqueous or nonaqueous isotonic sterile solutionor suspension.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive formulation in water-soluble form. Suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Aqueous injection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulary agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the preparations described previously, the formulationsmay also be formulated as a depot preparation. Such long actingformulations may be administered by intramuscular injection. Thus, forexample, the formulation may be formulated with suitable polymeric orhydrophobic materials, for example as an emulsion in an acceptable oil,or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Compositions and formulations of this invention may also be formulatedfor topical delivery and may be applied to the subject's skin using anysuitable process for application of topical delivery vehicle. Forexample, the formulation may be applied manually, using an applicator,or by a process that involves both. Following application, theformulation may be worked into the subject's skin, e.g., by rubbing.Application may be performed multiple times daily or on a once-dailybasis. For example, the formulation may be applied to a subject's skinonce a day, twice a day, or multiple times a day, or may be applied onceevery two days, once every three days, or about once every week, onceevery two weeks, or once every several weeks.

The formulations or pharmaceutical compositions described herein may beadministered to the subject by any suitable means. Examples of methodsof administration include, among others, (a) administration viainjection, subcutaneously, intraperitoneally, intravenously,intramuscularly, intradermally, intraorbitally, intracapsularly,intraspinally, intrasternally, or the like, including infusion pumpdelivery; (b) administration locally such as by injection directly inthe renal or cardiac area, e.g., by depot implantation; as well asdeemed appropriate by those of skill in the art for bringing the activecompound into contact with living tissue.

The exact formulation, route of administration and dosage for thepharmaceutical compositions can be chosen by the individual physician inview of the patient's condition. See, e.g., Goodman & Gilman's ThePharmacological Basis of Therapeutics, 12^(th) Ed., Sec. 1, 2011.Typically, the dose range of the composition administered to the patientcan be from about 0.5 to about 1000 mg/kg of the patient's body weight.The dosage may be a single one or a series of two or more given in thecourse of one or more days, as is needed by the patient. In instanceswhere human dosages for compounds have been established for at leastsome condition, the dosages will be about the same, or dosages that areabout 0.1% to about 500%, more preferably about 25% to about 250% of theestablished human dosage. Where no human dosage is established, as willbe the case for newly-discovered pharmaceutical compositions, a suitablehuman dosage can be inferred from ED50 or ID50 values, or otherappropriate values derived from in vitro or in vivo studies, asqualified by toxicity studies and efficacy studies in animals.

EXAMPLES Example 1 Preparation of siRNA Lipid Nanoparticle DrugFormulations by Lyophilization and Reconstitution

Lipid nanoparticles were synthesized with high speed injection oflipid/ethanol solution into an siRNA buffer solution for about 10minutes. Afterward, a second buffer, selected from citrate buffer pH6.1, PBS pH 7.0, Tris pH 7.2, and HEPES pH 7.4, was diafiltered and usedas the external buffer through TFF cartridges to make a final productaqueous suspension.

Various amounts of protectant compounds were added to the final productaqueous suspension, followed by 0.2/0.8 micrometer filtration. Lipidnanoparticles were prepared in either 500 mL or 1000 mL batches.

Results: It was surprisingly found that the lipid nanoparticles survivedthe lyophilization process, and that the lyophile provided areconstituted suspension of lipid nanoparticles having an averageparticle size close to the size that was present in the originalsuspension.

Example 2 Reconstituted siRNA Drug Formulations of this InventionExhibit Stable Particle Size and siRNA Encapsulation, which are Suitablefor Use in Drug Products. The Surprising Level of Stability of the siRNAFormulations of this Invention Arises from the Properties of theProtected Lyophilization Composition

In this study, a siRNA targeted to Hsp47 was formulated in liposomalnanoparticles with the following approximate composition: ionizablelipids, 40 mol %, DOPE, 30 mol %, Cholesterol, 25 mol %, and PEG-DMPE,5.0 mol %.

The nanoparticle formulations were lyophilized with a protectantcomposition containing sucrose and (2-hydroxypropyl)-β-cyclodextrin. Thetotal protectant content of the composition was 10% (w/v). Thequantities used were 6% sucrose and 4%(2-hydroxypropyl)-β-cyclodextrin). Thus, the content of(2-hydroxypropyl)-β-cyclodextrin was 40% (w/v) of the total amount ofsucrose plus (2-hydroxypropyl)-β-cyclodextrin.

Lipid nanoparticles were synthesized by high speed injection oflipid/ethanol solution into an siRNA buffer solution, to make a finalproduct aqueous suspension. The initial bulk formulation of thesiRNA-containing nanoparticles had an average nanoparticle size range of99-101 nm.

The lyophilized, reconstituted siRNA drug formulations were tested forstability of particle size and encapsulation of the siRNA.

In general, it is most preferred that the siRNA nanoparticleformulations exhibit less than about 10% change in average particle sizebetween the pre-lyophilized state and the lyophilized, reconstitutedstate. Further, it is most preferred that the siRNA nanoparticleformulations exhibit at least about 85% siRNA encapsulation efficiencyof the lyophilized, reconstituted state.

The stability of the reconstituted siRNA nanoparticle products are shownin Table 2. In Table 2, the average particle size and siRNAencapsulation efficiency after lyophilization (AL) and reconstitutionare shown along with similar results obtained from a frozen, thawedsolution before lyophilization (BL).

TABLE 2 Nanoparticle stability before lyophilization and afterreconstitution Total protectant Z(avg) (nm) PDI Zeta (mV) % EE [siRNA] %(w/v) BL AL BL AL BL AL BL AL mg/mL  10% (A)* 105 108 0.140 0.167 −1.4−1.7 91 86 1.9 10% (B) 106 110 0.156 0.138 −1.8 −1.7 92 87 2.0 10% (C)106 108 0.150 0.165 −2.0 −1.7 92 87 1.9 10% (D) 105 111 0.146 0.157 −1.7−2.4 92 85 1.8 10% (A) 106 111 0.134 0.149 −1.5 −1.1 93 88 3.7 12.5%(A)  106 112 0.165 0.186 −1.7 −1.6 93 87 3.7 15% (A) 105 112 0.170 0.141−1.3 −1.1 93 87 3.6 15% (A) 106 116 0.140 0.149 −1.3 −0.5 93 88 4.4 15%(B) 105 116 0.154 0.157 −1.3 −0.7 93 88 4.4 15% (C) 105 118 0.151 0.150−1.1 −0.8 93 88 4.5 *In Table 2, (2-hydroxypropyl)-β-cyclodextrin)protectant compound from four different commercial sources A, B, C and Dwere used.

In Table 2, all protectant compositions exhibited suitable stability ofthe final reconstituted siRNA drug formulations. Except for samples atthe highest levels of siRNA concentration and total protectant (15%),the siRNA nanoparticle formulations exhibited less than 10% change inaverage particle size between the pre-lyophilized state and thelyophilized, reconstituted state, as well as at least 85% siRNAencapsulation efficiency of the lyophilized, reconstituted state.

The results in Table 2 show that reconstituted siRNA drug formulationsof this invention that were prepared from nanoparticle formulations bylyophilization from a protectant composition containing 60% sucrose and40% (2-hydroxypropyl)-β-cyclodextrin were advantageously stable inaverage particle size and siRNA encapsulation efficiency.

Example 3 Dimethylnitrosamine (DMN) Induced Liver Fibrosis Rat Model

Reconstituted siRNA drug formulations of this invention exhibitedprofound and surprising potency for gene silencing in vivo. In vivoknockdown with lyophilized and reconstituted siRNA formulations wasobserved. siRNAs encapsulated in a liposomal formulation were used in aDimethylnitrosamine (DMN) Induced Liver Fibrosis Rat Model.

A siRNA targeted to Hsp47 (GP46) was formulated in liposomalnanoparticles with the following approximate composition: ionizablelipids, 40 mol %, DOPE, 30 mol %, Cholesterol, 25 mol %, and PEG-DMPE,5.0 mol %.

The nanoparticle formulations were lyophilized with a protectantcomposition containing sucrose and (2-hydroxypropyl)-β-cyclodextrin. Thetotal protectant content was 10% (w/v). The content of(2-hydroxypropyl)-β-cyclodextrin was varied from 20% to 40% (w/v).

The samples were obtained as a fresh, same day lyophilized cake storedat −80 C. The samples were reconstituted with saline and further dilutedwith saline to a concentration of 0.17 mg/mL siRNA. Reconstitution timewas about 20 s with 3 mL volume.

The final drug product reconstituted solutions of the Hsp47 siRNA weretested for in vivo potency, which is a rigorous test for the viabilityof lyophilized, reconstituted nanoparticles containing a nucleic acidagent.

Naïve Sprague Dawley rats in ten groups of 7-8 males with weight range180-200 g were used in this study. A liquid dosage form with PBS at pH7.4 was used. On the dosing day, prior to administration, formulationwas reconstituted and diluted using saline into concentrations by group.The frozen control formulation was thawed and diluted one day beforeadministration. An amount of DMN to achieve 5 mg/mL of clear dosingsolution on the day of injection was added to PBS at pH 7.4.Administration was by intraperitoneal injection. Dosing was QD, Day 1-3,for 3 consecutive days. Dose was 0.5 to 1.5 mg/kg (siRNA) usingformulation concentration range of 0.17-0.5 mg/mL, with administeredvolume 3 mL/kg. Rats were weighted before DMN administration and animalswere injected on day 1-3 by intraperitoneal of 10 mg/kg of DMN (solutionat 5 mg/ml), with dosing volume 2 mL/kg. On day 4-6, animals wereinjected with DMN with dosing volume 1 mL/kg. On day 5, DMN-treatedanimals were randomized into groups based on body weight (day 5) beforethe drug administration. Test article was administrated on Day 6 (thefirst day of DMN injection was day 1). On day 7, rat livers wereobtained and immediately flushed with PBS, pH 7.4 (40 mL at a rate of 20mL/min) through clipped hepatic portal vein. One 2 mm thick transverseliver section was collected from the left lateral lobs.

gp46 mRNA knockdown evaluation. Total RNA from rat liver was extractedusing RNeasy columns (Qiagen). RNA was quantified using a Nanodropspectrophotometer.

As shown in FIG. 1, the reconstituted siRNA drug formulation of thisinvention that was protected with 40% (2-hydroxypropyl)-β-cyclodextrinexhibited profound and surprising potency for gene silencing of Hsp47(GP46) in vivo.

In particular, the in vivo Hsp47 (GP46) gene silencing potency of theformulation protected with 40% (2-hydroxypropyl)-β-cyclodextrin wasessentially 100%.

To the contrary, the in vivo potency of formulations containing 20% to30% (2-hydroxypropyl)-β-cyclodextrin exhibited unacceptably low geneknockdown, being only 47% and 32%, respectively.

In sum, the unexpectedly advantageous result shows that protectantcompositions for lyophilization of liposomal siRNA formulations of thisinvention can be made with at least 40% (2-hydroxypropyl)-β-cyclodextrinin a composition containing sucrose and(2-hydroxypropyl)-β-cyclodextrin.

Physical characterization showed that reconstituted siRNA drugformulations of this invention that were prepared from nanoparticleformulations by lyophilization from a protectant composition containingfrom about 40% to about 70% (2-hydroxypropyl)-β-cyclodextrin, and theremainder sucrose, were advantageously stable in average particle size.Below about 40% (2-hydroxypropyl)-3-cyclodextrin, the formulationstended to have anomalously increased encapsulation values, which is anindication of unwanted structural changes. Thus, the preferred range forthe (2-hydroxypropyl)-β-cyclodextrin component was from about 40% toabout 70%.

In conclusion, a reconstituted siRNA drug formulation of this inventionutilizes from 40% to 70% (2-hydroxypropyl)-β-cyclodextrin withsurprising potency for a nucleic acid drug agent in vivo.

Example 4 Reconstituted siRNA Drug Formulations of this InventionExhibited Sufficient Plasma Concentration for Gene Silencing In Vivo.Plasma Pharmacokinetics of Lyophilized and Reconstituted siRNAFormulations was Observed In Vivo

A siRNA targeted to Hsp47 (GP46) was formulated in liposomalnanoparticles with the following approximate composition: ionizablelipids, 40 mol %, DOPE, 30 mol %, Cholesterol, 25 mol %, and PEG-DMPE,5.0 mol %.

The nanoparticle formulations were lyophilized with a protectantcomposition containing sucrose and (2-hydroxypropyl)-β-cyclodextrin. Thetotal protectant content was 12.5% (w/v). The content of(2-hydroxypropyl)-β-cyclodextrin was 40% (w/v) of the total protectant,the remainder sucrose.

Plasma PK profiles were evaluated in Sprague Dawley rats following anintravenous administration at a single dose level of the lyophilizedformulation compared to a frozen formulation. siRNA concentrations inplasma samples were determined by a hybridization-based ELISA method.Sprague-Dawley rats were prepared with a double jugular vein catheter.Animals were given a single bolus intravenous dose injection of the testmaterial via one jugular vein catheter over 15 seconds at Day 1.Approximately 0.30 mL of whole blood was collected from the jugular veincatheter of each animal into K2EDTA tubes at each time point.

As shown in FIG. 2, the plasma concentration pharmacokinetics of thelyophilized, reconstituted siRNA drug formulation was essentially thesame as a comparative control formulation that had only been frozen. Thelyophilized, reconstituted siRNA nucleic acid drug formulation providedsurprisingly efficient levels of drug agent in plasma.

For this experiment, the area under the time-concentration curve (AUC)and the peak plasma concentration (Cmax) after a single dose are shownin Table 3.

TABLE 3 Plasma pharmacokinetics for lyophilized, reconstituted nucleicacid formulations Reconstituted Frozen AUC 2751 2683 Cmax 2922 3350

In conclusion, this experiment shows that the plasma concentrationpharmacokinetics of a lyophilized, reconstituted nucleic acid drugformulation was essentially the same as a comparative positive controlformulation that had only been frozen. Thus, the lyophilized,reconstituted siRNA nucleic acid drug formulation provided surprisinglyefficient levels of drug agent in plasma, and was not degraded relativeto a non-lyophilized composition.

Example 5 Protecting Lipid Nanoparticles in the 100 nm Size Range

Lipid nanoparticles were synthesized with dispersion of lipid/ethanolsolution into a siRNA buffer, to make a final product aqueoussuspension. The nanoparticles had an average size of 105-106 nm. Thenanoparticles were synthesized using compound HEDC as an ionizable lipid(see, e.g. US 2013/022665 A1). The nanoparticles encapsulated a siRNAtargeted to Hsp47.

Table 4 shows the nanoparticle characteristics before lyophilization,where the final product aqueous suspension was merely frozen, thenthawed. Table 5 shows the nanoparticle characteristics afterlyophilization.

The increase in average particle size from Table 4 to Table 5 is only6.7%.

TABLE 4 Nanoparticle characteristics before lyophilization (100 nm sizerange) Sucrose/ Pre-lyophilization (Frozen) 2HPBCD Z(avg) Zeta EE[siRNA] yield No. (wt %/wt %) (nm) PDI (mV) (%) (mg/mL) (%) 1 6/4 1050.14 −1.4 91 2.1 103 2 6/4 106 0.16 −1.8 92 2.1 105 3 6/4 106 0.15 −2.092 2.1 104 4 6/4 105 0.15 −1.7 92 2.1 104 5 6/4 106 0.14 −1.3 93 5.1 1016 6/4 105 0.15 −1.3 93 4.9 99 7 6/4 105 0.15 −1.1 93 5.2 103 8 6/4 1060.16 −0.9 93 5.2 104

TABLE 5 Nanoparticle characteristics after lyophilization (100 nm sizerange) Sucrose/ Post-lyophilization (Reconstituted) 2HPBCD Z(avg) ZetaEE [siRNA] yield No. (wt %/wt %) (nm) PDI (mV) (%) (mg/mL) (%) 1 6/4 1080.17 −1.7 86 1.9 94 2 6/4 110 0.14 −1.7 87 2.0 98 3 6/4 108 0.16 −1.7 871.9 95 4 6/4 111 0.16 −2.4 85 1.8 92 5 6/4 116 0.15 −0.5 88 4.4 88 6 6/4116 0.16 −0.7 88 4.4 89 7 6/4 118 0.15 −0.8 88 4.5 90 8 6/4 114 0.12−1.3 87 4.42 88

In an additional test, nine final product solutions containing 6% (w/v)sucrose, 4% (w/v) (2-hydroxypropyl)-β-cyclodextrin, and a concentrationof a siRNA targeted to Hsp47 of 2 mg/mL were tested for average particlesize increase upon lyophilization and reconstitution. Afterlyophilization, the reconstituted drug products showed surprisinglylittle increase in average particle size of less than 5%, from 102 nm to107 nm, as compared to the final product solutions beforelyophilization.

Example 6 Protecting Lipid Nanoparticles in the 50 nm Size Range

Lipid nanoparticles were synthesized with high speed injection oflipid/ethanol solution into an siRNA buffer, to make a final productaqueous suspension. The nanoparticles had an average size of 48-50 nm.The nanoparticles were synthesized using Compound A6 as an ionizablelipid. The nanoparticles encapsulated a siRNA targeted to Hsp47 at 2mg/mL.

Table 6 shows the nanoparticle characteristics before lyophilization(BL) and after lyophilization (AL) of nanoparticles in the 50 nm range.

TABLE 6 Nanoparticle characteristics before and after lyophilization ofnanoparticles in the 50 nm range Sucrose/2HPBCD Z(avg) (nm) PDI Zeta(mV) % EE (wt %/wt %) BL AL BL AL BL AL BL AL 5/5 48 53 0.07 0.13 −2.3−3.3 76 66 6/4 48 51 0.07 0.14 −2.8 −4.8 81 68 12/0  50 75 0.08 0.25−4.9 −2.0 86 81

Example 7 Protecting siRNA Lipid Nanoparticles for Long Term Storage

Reconstituted siRNA drug formulations of this invention exhibited longterm stability for gene silencing in vivo.

A siRNA targeted to Hsp47 (GP46) was formulated in liposomalnanoparticles with the following approximate composition: ionizablelipids, 40 mol %, DOPE, 30 mol %, Cholesterol, 25 mol %, and PEG-DMPE,5.0 mol %.

The nanoparticle formulations were lyophilized with a protectantcomposition containing sucrose and (2-hydroxypropyl)-β-cyclodextrin. Thetotal protectant content was from 10% to 12.5% to 15% (w/v). The contentof (2-hydroxypropyl)-β-cyclodextrin was 40% (w/v), sucrose 60%. Thecyclodextrin was CAVITRON W7 HP5 PHARMA cyclodextrin.

The vials were stored at temperatures shown in Table 7 for 4 weeks.After storage and reconstitution, the average size of the nanoparticles(PS, Z-avg) was surprisingly stable, as shown in Table 7.

As shown in Table 7, the size of the siRNA nanoparticles was within 4%of their size in the original composition.

TABLE 7 Nanoparticle characteristics Sample Temperature −20 C.Temperature 5 C. total % Initial Z-avg 1 mo Z-avg Initial Z-avg 1 moZ-avg protectant Z-avg PDI Z-avg PDI Z-avg PDI Z-avg PDI 10 103 0.12 990.15 103 0.12 102 0.14 12.5 101 0.13 101  0.15 101 0.13 100 0.16 15 1020.13 — — 102 0.13 102 0.15 10 100 0.13 99 0.17 100 0.13 96 0.16 12.5 950.16 94 0.14 95 0.16 94 0.14 15 96 0.13 95 0.13 96 0.13 96 0.13 10 950.15 95 0.16 95 0.15 96 0.15 12.5 95 0.15 92 0.11 95 0.15 93 0.14 15 940.13 92 0.14 94 0.13 91 0.14

Example 8 Protecting siRNA Lipid Nanoparticles for Long Term Storage

Reconstituted siRNA drug formulations of this invention exhibited longterm stability for gene silencing in vivo.

A siRNA targeted to Hsp47 (GP46) was formulated in liposomalnanoparticles with the following approximate composition: ionizablelipids, 40 mol %, DOPE, 30 mol %, Cholesterol, 25 mol %, and PEG-DMPE,5.0 mol %.

The nanoparticle formulations were lyophilized with a protectantcomposition containing sucrose and (2-hydroxypropyl)-β-cyclodextrin. Thetotal protectant content was from 10% to 12.5% to 15% (w/v). The contentof (2-hydroxypropyl)-β-cyclodextrin was 40% (w/v), sucrose 60%. Thecyclodextrin was CAVITRON W7 HP7 PHARMA cyclodextrin.

The vials were stored at temperatures shown in Table 8 for 4 weeks.After storage and reconstitution, the average size of the nanoparticles(PS, Z-avg) was surprisingly stable, as shown in Table 8.

As shown in Table 8, the size of the siRNA nanoparticles was within 5%of their size in the original composition.

TABLE 8 Nanoparticle characteristics Sample Temperature −20 C.Temperature 5 C. total % Initial Z-avg 1 mo Z-avg Initial Z-avg 1 moZ-avg protectant Z-avg PDI Z-avg PDI Z-avg PDI Z-avg PDI 10 103 0.11 990.14 103 0.11 99 0.15 12.5 101 0.14 99 0.15 101 0.14 98 0.15 15 102 0.15— — 102 0.15 97 0.14 10 96 0.13 95 0.13 96 0.13 93 0.13 12.5 94 0.12 910.12 94 0.12 91 0.13 15 94 0.13 89 0.14 94 0.13 91 0.14 10 94 0.15 890.15 94 0.15 89 0.13 12.5 91 0.15 87 0.13 91 0.15 88 0.14 15 90 0.14 860.15 90 0.14 87 0.12

All publications, patents and literature specifically mentioned hereinare incorporated by reference in their entirety for all purposes.

It is understood that this invention is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention. It will be readilyapparent to one skilled in the art that varying substitutions andmodifications can be made to the description disclosed herein withoutdeparting from the scope and spirit of the description, and that thoseembodiments are within the scope of this description and the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprises,” “comprising”,“containing,” “including”, and “having” can be used interchangeably, andshall be read expansively and without limitation.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For Markush groups, those skilled in theart will recognize that this description includes the individualmembers, as well as subgroups of the members of the Markush group.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose.

What is claimed is:
 1. A composition for making a solid lyophile oflipid nanoparticles comprising one or more nucleic acid active agents,the composition comprising: an aqueous suspension of the lipidnanoparticles in a pharmaceutically acceptable solution, wherein thelipid nanoparticles encapsulate the one or more nucleic acid activeagents; a dextrin compound; and a saccharide sugar compound.
 2. Thecomposition of claim 1, wherein the total amount of the dextrin andsugar compounds is from 2% to 20% (w/v) of the composition.
 3. Thecomposition of claim 1, wherein the dextrin compound is from 40% to 70%(w/v) of the total amount of the dextrin and sugar compounds.
 4. Thecomposition of claim 1, wherein the dextrin compound is from 40% to 55%(w/v) of the total amount of the dextrin and sugar compounds.
 5. Thecomposition of claim 1, wherein the dextrin compound is 40% to 45% (w/v)of the total amount of the dextrin and sugar compounds.
 6. Thecomposition of claim 1, wherein upon lyophilization and reconstitutionof the composition, the average size of the nanoparticles is within 10%of their size in the original composition.
 7. The composition of claim1, wherein upon lyophilization, storage and reconstitution of thecomposition, the average size of the nanoparticles is within 10% oftheir size in the original composition.
 8. The composition of claim 7,wherein the lyophilized composition is stored at 5° C. for at least onemonth.
 9. The composition of claim 7, wherein the lyophilizedcomposition is stored at −20° C. for at least one month.
 10. Thecomposition of claim 1, wherein the nanoparticles have an averagediameter of from 45 nm to 110 nm.
 11. The composition of claim 1,wherein the concentration of the nucleic acid active agents is from 1mg/mL to 10 mg/mL.
 12. The composition of claim 1, wherein the lipidnanoparticles comprise a compound selected from compound A6, compoundA9, compound AA, compound AB, compound C2, compound F5, compound F7, andcompound C24.
 13. The composition of claim 1, wherein the one or morenucleic acid active agents are RNAi molecules capable of mediating RNAinterference.
 14. The composition of claim 13, wherein the RNAimolecules are siRNAs, shRNAs, ddRNAs, piRNAs, or rasiRNAs.
 15. Thecomposition of claim 1, wherein the one or more nucleic acid activeagents are miRNAs, antisense RNAs, plasmids, hybrid oligonucleotides, oraptamers.
 16. The composition of claim 1, wherein the pharmaceuticallyacceptable solution is a HEPES buffer, a phosphate buffer, a citratebuffer, or a buffer containing Tris(hydroxymethyl)aminomethane.
 17. Thecomposition of claim 1, wherein the dextrin compound is a cyclodextrin.18. The composition of claim 17, wherein the cyclodextrin compound hasone or more of the 2, 3 and 6 hydroxyl positions substituted withsulfoalkyl, benzenesulfoalkyl, acetoalkyl, hydroxyalkyl, hydroxyalkylsuccinate, hydroxyalkyl malonate, hydroxyalkyl glutarate, hydroxyalkyladipate, hydroxyalkyl, hydroxyalkyl maleate, hydroxyalkyl oxalate,hydroxyalkyl fumarate, hydroxyalkyl citrate, hydroxyalkyl tartrate,hydroxyalkyl malate, or hydroxyalkyl citraconate groups.
 19. Thecomposition of claim 17, wherein the cyclodextrin compound is(2-hydroxypropyl)-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrinsuccinate, (2-hydroxypropyl)-γ-cyclodextrin, or2-hydroxypropyl-γ-cyclodextrin succinate.
 20. The composition of claim17, wherein the cyclodextrin compound is sulfobutyl ether β-cyclodextrinor sulfobutyl ether γ-cyclodextrin.
 21. The composition of claim 17,wherein the cyclodextrin compound is methyl-β-cyclodextrin ormethyl-γ-cyclodextrin.
 22. The composition of claim 17, wherein thecyclodextrin compound is attached to a polymer chain or network.
 23. Thecomposition of claim 17, wherein the cyclodextrin compound includes anadsorbate compound.
 24. The composition of claim 23, wherein theadsorbate compound is selected from cholesterol, lanosterol, zymosterol,zymostenol, desmosterol, stigmastanol, dihydrolanosterol,7-dehydrocholesterol, pegylated cholesterol, cholesteryl acetate,cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate,cholesteryl myristate, cholesteryl palmitate, cholesteryl behenate,cholesteryl stearate, cholesteryl caprylate, cholesteryl n-decanoate,cholesteryl dodecanoate, cholesteryl nervonate, cholesteryl pelargonate,cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate,cholesteryl erucate, cholesteryl heptanoate, cholesteryl linolelaidate,cholesteryl linoleate, beta-sitosterol, campesterol, ergosterol,brassicasterol, delta-7-stigmasterol, and delta-7-avenasterol.
 25. Thecomposition of claim 1, wherein the saccharide sugar compound is amonosaccharide or disaccharide sugar compound.
 26. The composition ofclaim 25, wherein the sugar compound is selected from sucrose, lactose,lactulose, maltose, trehalose, cellobiose, kojibiose, sakebiose,isomaltose, sophorose, laminaribiose, gentiobiose, turanose, maltulose,isomaltulose, gentiobiulose, mannobiose, melibiose, melibiulose, andxylobiose.
 27. A process for making a solid lyophile of one or morenucleic acid active agents, the process comprising lyophilizing acomposition according to claim
 1. 28. A solid lyophile made by theprocess of claim
 27. 29. A process for making a drug product comprisingreconstituting a solid lyophile according to claim
 27. 30. A drugproduct made by the process of claim
 29. 31. A process for making anucleic acid drug product, the process comprising: synthesizing lipidnanoparticles, wherein the lipid nanoparticles encapsulate one or morenucleic acid active agents; providing an aqueous suspension of the lipidnanoparticles in a pharmaceutically acceptable solution; adding adextrin compound to the solution containing the lipid nanoparticles;adding a saccharide sugar compound to the solution containing the lipidnanoparticles; lyophilizing the solution containing the lipidnanoparticles, thereby forming a solid lyophile; reconstituting thelyophile in a pharmaceutically acceptable carrier, thereby forming anucleic acid drug product.
 32. The process of claim 31, wherein thetotal amount of the dextrin and saccharide sugar compounds is from 2% to20% (w/v) of the solution containing the lipid nanoparticles.
 33. Theprocess of claim 31, wherein the dextrin compound is from 40% to 70%(w/v) of the total amount of the dextrin and saccharide sugar compounds.34. The process of claim 31, wherein the dextrin compound is from 40% to55% (w/v) of the total amount of the dextrin and saccharide sugarcompounds.
 35. The process of claim 31, wherein the dextrin compound is40% to 45% (w/v) of the total amount of the dextrin and saccharide sugarcompounds.
 36. The process of claim 31, wherein upon reconstitution, theaverage size of the nanoparticles is within 10% of their size whensynthesized.
 37. The process of claim 31, further comprising storing thelyophile before reconstitution.
 38. The process of claim 37, whereinupon storage and reconstitution of the lyophile, the average size of thenanoparticles is within 10% of their size when synthesized.
 39. Theprocess of claim 37, wherein the lyophile is stored at 5° C. for atleast one month.
 40. The process of claim 37, wherein the lyophile isstored at −20° C. for at least one month.
 41. The process of claim 31,wherein the nanoparticles have an average diameter of from 45 nm to 110nm.
 42. The process of claim 31, wherein the concentration of thenucleic acid active agents is from 1 mg/mL to 10 mg/mL.
 43. The processof claim 31, wherein the one or more nucleic acid active agents are RNAimolecules capable of mediating RNA interference.
 44. The process ofclaim 31, wherein the pharmaceutically acceptable solution is a HEPESbuffer, a phosphate buffer, a citrate buffer, or a buffer containingTris(hydroxymethyl)aminomethane.
 45. The process of claim 31, whereinthe dextrin compound is a cyclodextrin.
 46. The process of claim 45,wherein the cyclodextrin compound has one or more of the 2, 3 and 6hydroxyl positions substituted with sulfoalkyl, benzenesulfoalkyl,acetoalkyl, hydroxyalkyl, hydroxyalkyl succinate, hydroxyalkyl malonate,hydroxyalkyl glutarate, hydroxyalkyl adipate, hydroxyalkyl, hydroxyalkylmaleate, hydroxyalkyl oxalate, hydroxyalkyl fumarate, hydroxyalkylcitrate, hydroxyalkyl tartrate, hydroxyalkyl malate, or hydroxyalkylcitraconate groups.
 47. The process of claim 45, wherein thecyclodextrin compound is (2-hydroxypropyl)-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin succinate,(2-hydroxypropyl)-γ-cyclodextrin, or 2-hydroxypropyl-γ-cyclodextrinsuccinate.
 48. The process of claim 45, wherein the cyclodextrincompound is sulfobutyl ether β-cyclodextrin or sulfobutyl etherγ-cyclodextrin.
 49. The process of claim 45, wherein the cyclodextrincompound is methyl-β-cyclodextrin or methyl-γ-cyclodextrin.
 50. Theprocess of claim 45, wherein the cyclodextrin compound includes anadsorbate compound.
 51. The process of claim 31, wherein the saccharidesugar compound is a monosaccharide or disaccharide sugar compound. 52.The process of claim 31, wherein the pharmaceutically acceptable carrieris sterile water, water for injection, sterile normal saline,bacteriostatic water for injection, or a nebulizer solution.
 53. Theprocess of claim 31, wherein the pharmaceutically acceptable carrier isa pharmaceutically acceptable solution.
 54. The process of claim 31,wherein the reconstituted nucleic acid drug product has less 0.001%(w/v) of aggregate particles with a size greater than 0.2 um.
 55. Theprocess of claim 31, wherein the nucleic acid drug product isreconstituted in a time period of 3 to 30 seconds.
 56. The process ofclaim 31, wherein the nucleic acid drug product is reconstituted after astorage time period of six months and retains 80% activity of thenucleic acid agents.